RESUMEN
The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.
Asunto(s)
Sistemas CRISPR-Cas , Carcinoma de Células Escamosas , Transformación Celular Neoplásica , Evolución Clonal , Células Clonales , Análisis de la Célula Individual , Factores de Necrosis Tumoral , Animales , Femenino , Humanos , Masculino , Ratones , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Evolución Clonal/genética , Células Clonales/citología , Células Clonales/metabolismo , Células Clonales/patología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Sistemas CRISPR-Cas/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Macrófagos/metabolismo , Mutación , Invasividad Neoplásica/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/genética , Análisis de la Célula Individual/métodos , Transcriptoma/genética , Factores de Necrosis Tumoral/genética , Factores de Necrosis Tumoral/metabolismo , Comunicación Autocrina , Análisis de SupervivenciaRESUMEN
Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that microridges are formed of F-actin networks and require the function of the Arp2/3 complex for their maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.